diversity
Article
Statistical Assessment of Phenol Biodegradation by a
Metal-Tolerant Binary Consortium of Indigenous
Antarctic Bacteria
Kavilasni Subramaniam 1 , Siti Aqlima Ahmad 1,2 , Peter Convey 3,4 , Noor Azmi Shaharuddin 1 ,
Khalilah Abdul Khalil 5 , Tengku Athirrah Tengku-Mazuki 1 , Claudio Gomez-Fuentes 2,6
and Azham Zulkharnain 7, *






Citation: Subramaniam, K.;
Ahmad, S.A.; Convey, P.;
Shaharuddin, N.A.; Khalil, K.A.;
Tengku-Mazuki, T.A.;
Gomez-Fuentes, C.; Zulkharnain, A.
Statistical Assessment of Phenol
Biodegradation by a Metal-Tolerant
Binary Consortium of Indigenous
Antarctic Bacteria. Diversity 2021, 13,
643. https://doi.org/10.3390/
d13120643
Academic Editors:
Angelina Lo Giudice
and Carmen Rizzo
Received: 4 October 2021
Accepted: 24 November 2021
Published: 4 December 2021
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Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Diversity 2021, 13, 643. https://doi.org/10.3390/d13120643
1 Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia,
Serdang 43400, Selangor, Malaysia; kavilasnisubramaniam@gmail.com (K.S.); aqlima@upm.edu.my (S.A.A.);
noorazmi@upm.edu.my (N.A.S.); drathirrah@gmail.com (T.A.T.-M.)
2 Center for Research and Antarctic Environmental Monitoring (CIMAA), Universidad de Magallanes, Avda.
Bulnes, Punta Arenas 01855, Región de Magallanes y Antártica Chilena, Chile; claudio.gomez@umag.cl
3 British Antarctic Survey, NERC, High Cross, Madingley Road, Cambridge CB3 0ET, UK; pcon@bas.ac.uk
4 Department of Zoology, University of Johannesburg, P.O. Box 524, Auckland Park 2006, South Africa
5 School of Biology, Faculty of Applied Sciences, Universiti Teknologi MARA,
Shah Alam 40450, Selangor, Malaysia; khali552@uitm.edu.my
6 Department of Chemical Engineering, Universidad de Magallanes, Avda. Bulnes,
Punta Arenas 01855, Región de Magallanes y Antártica Chilena, Chile
7 Department of Bioscience and Engineering, College of Systems Engineering and Science, Shibaura Institute of
Technology, 307 Fukasaku, Minuma-ku, Saitama 337-8570, Japan
* Correspondence: azham@shibaura-it.ac.jp
Abstract: Since the heroic age of Antarctic exploration, the continent has been pressurized by
multiple anthropogenic activities, today including research and tourism, which have led to the
emergence of phenol pollution. Natural attenuation rates are very slow in this region due to the harsh
environmental conditions; hence, biodegradation of phenol using native bacterial strains is recognized
as a sustainable remediation approach. The aim of this study was to analyze the effectiveness of
phenol degradation by a binary consortium of Antarctic soil bacteria, Arthrobacter sp. strain AQ5-06,
and Arthrobacter sp. strain AQ5-15. Phenol degradation by this co-culture was statistically optimized
using response surface methodology (RSM) and tolerance of exposure to different heavy metals
was investigated under optimized conditions. Analysis of variance of central composite design
(CCD) identified temperature as the most significant factor that affects phenol degradation by this
consortium, with the optimum temperature ranging from 12.50 to 13.75 ◦ C. This co-culture was
able to degrade up to 1.7 g/L of phenol within seven days and tolerated phenol concentration as
high as 1.9 g/L. Investigation of heavy metal tolerance revealed phenol biodegradation by this
co-culture was completed in the presence of arsenic (As), aluminum (Al), copper (Cu), zinc (Zn),
lead (Pb), cobalt (Co), chromium (Cr), and nickel (Ni) at concentrations of 1.0 ppm, but was inhibited
by cadmium (Cd), silver (Ag), and mercury (Hg).
Keywords: cold climate; pollution; statistical optimization; mixed culture; metal ion
1. Introduction
Antarctica and its surrounding Southern Ocean are widely considered to be among
the Earth’s last pristine wildernesses, compared with the rest of the world, and has only
recently been discovered and subsequently impacted by humans. The negotiation of the
Antarctic Treaty in 1959, which came into force in 1961, declared that this ‘white canvas’
was to be a land of science, research, and peaceful cooperation between nations. Since
then, the continent has seen inexorable growth in scientific exploration, numbers, and
extent of research stations, research activities, marine transportation and, more recently,
https://www.mdpi.com/journal/diversity
Diversity 2021, 13, 643 2 of 19

tourism [1,2]. Human presence and activity present a distinct set of threats to the Antarctic
environment, one of which is pollution [3,4]. Despite the strict guidelines provided in
the Protocol on Environmental Protection to the Antarctic Treaty to protect the Antarctic
environment, there remains potential for significant marine and terrestrial impacts at
various scales due to accidents such as shipwrecks and pollution associated with research
activities and research stations [2]. Historical and, in some cases, ongoing application of
inappropriate waste management practices have led to the release of contaminants both to
the surrounding terrestrial and near-shore aquatic environments [5–7].
The primary routes by which phenol has entered the Antarctic environment are
through oil spills and inappropriate waste management practices. The most recent major
oil spill in the marine environment took place in November 2007 when the cruise vessel, MS
Explorer, carrying 1200 L of petrol, 24,000 L of lubricant oil, and 190,000 L of marine gas oil
was holed by ice and sank soon after in the Bransfield Strait between the Antarctic Peninsula
and South Shetland Islands [8]. Further diesel spills have occurred at research stations,
such as at the Argentinian Carlini Station, King George Island, in 2009 [9]. Following
such spills, POPs, including phenol, can be transported more widely through physical
and chemical processes, resulting from the addition of dispersants, subsequent shoreline
erosion, water washing, or volatilization, while retaining their toxicity [10]. In addition
to catastrophic marine accidents, refueling operations for stations on land, pipeline faults
within the stations’ transport, and smaller-scale operations such as refueling of vehicles
are the sources of the majority of terrestrial fuel spills [11]. Phenol is one of the major
components used in the production of diesel fuel [12] and Singh et al. [13] reported that
petroleum wastewater containing 140–480 mg/L of oil included phenol at concentrations
ranging from 1.2 to 3.71 mg/L. Moreover, discharging lightly treated grey water into the
sea from coastal research stations and, historically, also onto ice and other parts of the local
environment from inland stations, can lead to phenol pollution [14], as phenol is one of the
main components in many household and pharmaceutical products [15,16]. Phenol and
phenol-derived pollutants such as vanillic acid, homovanilic acid, syringic acid, p-coumaric
acid, and polychlorinated biphenyls are persistent in the Antarctic environment due to the
continent’s frigid and often dry climate, limiting the rates of both biological processes and
abiotic degradation [17–19].
The hazardous effects of phenol, even at low concentrations in the environment, are
more than sufficient to justify mitigation. In recent decades, research into the treatment
of phenol pollution has gained momentum [20,21]. Remediation in polar regions has
traditionally relied upon excavation and removal of polluted soil, which may still be ap-
propriate in instances of severe contamination. However, this type of remediation has a
considerable direct impact on the Antarctic environment and can result in soil shrinkage,
land slumping, permafrost melt, or alteration of groundwater flow. Such approaches are
also expensive and logistically challenging [22]. Therefore, bioremediation using native
microorganisms has attractive potential as a sustainable approach for restoring contami-
nated sites. Polluted wastewater is often compounded by the presence of various heavy
metals as co-contaminants, which can have further adverse effects on the function and
availability of microorganisms [23–26]. Concordantly, traces of heavy metals were detected
in the wastewater discharged from various Antarctic research stations [27,28]. Thus, heavy
metal toxicity may compromise the biodegradation of phenol. Studies of the relationship
between bacterial tolerance to heavy metals and degradation of phenolic compounds are,
therefore, an important element of the development of bioremediation applications.
In recent years, studies using mixed microbial cultures have attracted increasing
research attention, especially recognizing the potential role of cooperative or synergistic
effects result from the possession of different but complementary biochemical degradation
pathways in the different strains present [29]. A range of studies are now available using
mixed culture methods, encouraging investigation of the application of this approach in
Antarctica using native microorganisms. Nevertheless, several limitations exist, such as
the limited capability of microbial species in the field and the absence of benchmark values
Diversity 2021, 13, 643 3 of 19

against which to test the technique’s effectiveness for extensive field application [30]. In
addition, the effectiveness of biodegradation depends on several external factors such
as temperature, pH, nutrients availability, salinity, and site characteristics [21,31]; hence,
investigation of effect of these factors is crucial to indicate the optimal conditions that play
a role in bioremediation. A statistical approach using design of experiment (DoE) tools is
efficient and versatile for the optimization of processes comprising multiple variables as it
reduces the number of experimental trials and identifies the influence of individual factors,
in addition to the interaction effects on the desired response [32,33]. Central composite
design (CCD) and response surface methodology (RSM) comprising factorial designs and
regression analyses are some of the DoE tools used to design such experiments, evaluate the
significant factors, and build models to reveal optimum conditions for the biodegradation
process [34].
The present study focuses on the statistical evaluation of phenol degradation by
a binary consortium of the Antarctic soil bacteria, Arthrobacter sp. strain AQ5-06, and
Arthrobacter sp. strain AQ5-15 in combination with investigation of the consortium’s
tolerance of exposure to different heavy metals.

2. Materials and Methods

2.1. Bacterial Culture, Maintenance and Media Preparation
Arthrobacter sp. strains AQ5-06 (KX946127) and AQ5-15 (MK744046) from King George
Island, which were previously isolated and identified as diesel and phenol-degrading
bacteria [35–37], and maintained in glycerol stock under −80 ◦ C at the Eco-Remediation
Technology Laboratory, Universiti Putra Malaysia, were used in this study. These two
strains were inoculated individually in nutrient broth (NB) and incubated at 10 ◦ C at
150 rpm for 96 h. Then 1 mL culture broth was transferred to degradation culture medium
containing 0.5 g/L phenol and incubated for 96 h. Repeated transfers were performed
several times under the same conditions as earlier to acclimatize the cultures. Minimal
salt medium (MSM) (pH 7.5) was prepared by adding (g/L) K2 HPO4 (0.4), KH2 PO4 (0.2),
NaCl (0.1), Mg2 SO4 , (0.1), (NH4 )2 SO4 (0.4), MnSO4 ·H2 O (0.01), Fe2 (SO4 )·H2 O (0.01), and
NaMoO4 ·H2 O (0.01). Phenol was added after autoclaving the medium at 121 ◦ C for 20 min.
The working quantity of phenol medium (PM) was 50 mL for all experiments conducted in
250 mL Erlenmeyer flasks.

2.2. Phenol Biodegradation

A binary consortium of strains AQ5-06 AQ5-15 with initial inoculum size of
OD600 nm ≈ 1.00 ± 0.10 was prepared by blending them to a final concentration of
10.0% (v/v) and inoculating them into the PM prior to incubation. Phenol medium without
bacterial inoculation served as a control sample. Phenol degradation was assessed using
the 4-aminoantipyrine assay following the American Public Health Association method
and analyzed by UV-vis spectrophotometry at 510 nm [38]. Optical density at 600 nm was
used as a proxy measure of bacterial growth [39]. In brief, 1 mL of the culture medium
was centrifuged at 12,000× g for 15 min. The supernatant was separated from the pellet
and further used in the 4-AAP assay, while the pellet was diluted with 1 mL sterile dis-
tilled water and used to measure bacterial growth. All experiments were carried out in
triplicate in a batch shake flask. The percentage of phenol degradation was calculated
using Equation (1):

x2 − x1
Phenol degradation (%) = × 100% (1)
x2

where x2 is the initial phenol concentration and x1 is the residual phenol concentration.

2.3. Design of Experiment (DoE) by Response Surface Methodology (RSM)

The central composite design (CCD)-based RSM is a statistical tool widely used for
investigational design, modeling, and optimization. In this study, the experimental design
Diversity 2021, 13, 643 4 of 19

and statistical analyses were generated using Design-Expert® version 6.0.8 (Stat-Ease Inc.
Minneapolis, MN, USA) to optimize the culture conditions. In total, 30 runs with four
significant variables were considered at five different levels (−2, −1, 0, +1, +2) containing
16 factorial points, eight axial points, and six replications at the center point (Table 1). The
experimental values’ range of each factor was selected based on the optimum range of pure
culture. Phenol degradation was used as the response and the significance of the model
and each coefficient in the equation were examined using Fischer’s F-test and ANOVA.
Upon completion of DoE, biodegradation of phenol was carried out in the batch culture
to obtain an appropriate model. The model was validated by comparing the predicted
response with experimental data.

Table 1. Experimental ranges of four different variables tested in CCD.

Experimental Values
Variables Symbol
−2 −1 0 +1 +2
(NH4)2SO4 concentration (g/L) A 0.2 0.3 0.4 0.5 0.6
NaCl concentration (g/L) B 0.08 0.10 0.13 0.15 0.17
pH C 6.75 7.00 7.25 7.50 7.75
Temperature (◦ C) D 7.0 10.0 12.5 15.0 17.50

2.4. Heavy Metal Tolerance

Tolerance of exposure to the heavy metals arsenic (As), aluminum (Al), silver (Ag),
copper (Cu), cobalt (Co), chromium (Cr), cadmium (Cd), lead (Pb), nickel (Ni), zinc (Zn),
and mercury (Hg) was evaluated separately at a concentration of 1.0 ppm in MSM con-
taining 0.5 g/L phenol. Pure cultures of strain AQ5-06, strain AQ5-15, and the binary
consortium were inoculated individually and incubated under optimized conditions at
150 rpm. A quantity of 10.0% (v/v) of bacterial strain was inoculated into MSM in the
absence of heavy metals to act as control [40]. Data analysis for dose-response inhibition
was performed using GraphPad Prism version 9.0.2 and relative inhibition was plotted vs.
the heavy metal concentration and fitted to a standard inhibition dose-response curve to
generate a half maximal inhibitory concentration (IC50 ) value [41].

3. Results and Discussion

3.1. Statistical Optimization of Phenol Degradation Using RSM
Prior to statistical analysis, the effect of inoculum ratio of the two strains was de-
termined by adding, to 50 mL MSM containing 0.5 g/L of phenol, the acclimatized cell
cultures at varying amounts in such a way as to obtain AQ5-06:AQ5-15 ratios of 1:1, 1:3,
and 3:1. In each of the three cases, there was no apparent lag, but a better growth was
observed when the inoculum ratio was 1:1 (Table 2). Consortia with inoculum ratios of 1:3
and 3:1 have both slower growth and degradation rates. In agreement, Tecon and Or [42]
also reported that strains mixed in equal fractions are expected to have optimal mutualistic
interaction. The active performance of the selected binary consortium was confirmed
by comparing the outcome with the control sample. Pure culture of strain AQ5-06 was
reported to completely degrade 0.5 g/L of phenol within 120 h [35], and strain AQ5-15 to
completely degrade 0.5 g/L of phenol within 108 h [36]. As shown in Table 2, these two
strains performed better as the co-culture favored convergence of 1:1 partner ratio.

Table 2. Time taken for binary consortium with different inoculum ratios to completely degrade
0.5 g/L phenol at 10 ◦ C.

DMC Inoculum Ratio Growth Rate (h−1 ) Incubation Time (h)

1:1 0.0191 (±0.0263) 48
AQ5-06 + AQ5-15 1:3 0.0106 (±0.0017) 84
3:1 0.0121 (±0.0227) 84
Diversity 2021, 13, 643 5 of 19

The investigational results of phenol biodegradation by the binary consortium were

examined through RSM to generate an empirical model. Based on these outcomes, the rela-
tionship between the response and independent variables was identified and is described
by the second-order polynomial equation (Equation (2)):
Y = +94.78 − 0.022x1 − 1.47x2 + 1.26x3 + 8.12x4 − 4.45x1 2 − 4.67x2 2 − 4.59x3 2 − 13.12x4 2 (2)
where Y is the phenol degradation percentage, x1 , x2 , x3 , and x4 are the corresponding
independent variables (NH4 )2 SO4 concentration (g/L), NaCl concentration (g/L), pH, and
temperature (◦ C), respectively. These four factors were employed based on the RSM analy-
sis undertaken in previous studies on pure cultures of strains AQ5-06 and AQ5-15 [43,44].
Clarke [45] noted that a well-balanced medium comprising carbon and nitrogen is essential
for cell growth and maintenance, and Varjani and Upasani [46] found that nutrient avail-
ability, especially nitrogen demand, is crucial in hydrocarbons’ biodegradation. A tolerable
level of NaCl has a positive effect on the biological performance of microorganisms and ex-
ceeding that level can negatively affect the cell numbers and distribution, thus reducing the
rate of microbial metabolism [47]. Most microbes have lower tolerance to high or low pH
due to the significant effect on the biochemical reactions required for biodegradation [48].
Finally, temperature has a direct influence on the microbial growth rates and metabolic
processes, and can affect the physical state of pollutants [46].
Table 3 shows the ANOVA results of CCD on phenol degradation by the binary
consortium. The overall model was well supported and there is significant correlation
between the factors and response. One linear term D, and all four quadratic terms A2 , B2 ,
C2 , and D2 , were significant for phenol degradation. This indicated that the temperature
has the most significant effect on phenol degradation by this binary consortium. Although
the linear terms A, B, and C were not significant, significant second orders of these factors
indicated that they affect the response. The insignificant “lack of fit” value and high
coefficient of determination (R2 ) and adjusted R2 values confirm the model provides an
appropriate fit to the experimental data.

Table 3. Analysis of variance (ANOVA) of phenol degradation by the binary consortium.

Source Sum of Squares DF Mean Square F-Value Prob > F

Model 6879.31 8 859.91 37.51 <0.0001 ***
A 0.01 1 0.012 5.3 × 10−3 0.9819
B 51.92 1 51.92 2.26 0.1472
C 38.35 1 38.35 1.67 0.2099
D 1581.13 1 1581.13 68.97 <0.0001 ***
A2 544.17 1 544.17 23.74 <0.0001 ***
B2 598.61 1 598.61 26.11 <0.0001 ***
C2 577.34 1 577.34 25.18 <0.0001 ***
D2 4718.10 1 4718.10 205.80 <0.0001 ***
Residual 481.43 21 22.93
Lack of Fit 378.55 16 23.66 1.15 0.4773
Pure Error 102.88 5 20.58
Cor Total 7360.74 29
Standard deviation 4.79 R2 0.9346
Mean 73.31 Adjusted R2 0.9097
Coefficient variance 6.53 Predicted R2 0.7573
PRESS 1786.11 Adequate Precision 26.1944
A: Ammonium sulphate concentration (g/L); B: Sodium chloride concentration (g/L); C: pH; D: Temperature (◦ C). *** p < 0.001.

Figure 1a indicates the close similarity between the predicted and experimentally
achieved value for phenol degradation by the binary consortium, and Figure 1b reveals no
indication of non-normality, confirming the quadratic model is satisfactory for the purpose
of this analysis.
 PRESS 1786.11 Adequate Precision 26.1944
A: Ammonium sulphate concentration (g/L); B: Sodium chloride concentration (g/L); C: pH; D: Temperature (°C). *** p < 0.001.

Figure 1a indicates the close similarity between the predicted and experimentally
Diversity 2021, 13, 643 achieved value for phenol degradation by the binary consortium, and Figure 1b reveals6 of 19
no indication of non-normality, confirming the quadratic model is satisfactory for the pur-
pose of this analysis.

(a)

(b)
vv

Figure1.1. Diagnostic
Figure Diagnostic plots
plots of
of the
thecentral
centralcomposite
compositedesign
designmodel
modelofof
phenol degradation
phenol by by
degradation the the
binary consortium. (a) Similarity plot between predicted and actual values for phenol degradation;
binary consortium. (a) Similarity plot between predicted and actual values for phenol degradation;
(b) Normal probability plot against residuals for phenol degradation.
(b) Normal probability plot against residuals for phenol degradation.

Thehighly
The highlysignificant
significantinfluence
influenceofofthe
thetemperature
temperatureon onphenol
phenoldegradation
degradationisisshown
shown
ininFigure
Figure2.2.This
Thisindicated
indicatedthat
thateven
evenaasmall
smalldeviation
deviationinintemperature
temperaturecan
canhave
haveaadispro-
dispro-
portionate effect on phenol degradation achieved by the binary consortium. Temperature
portionate effect on phenol degradation achieved by the binary consortium. Temperature
is a crucial variable, especially in the polar regions. The optimum temperature for phenol
degradation by this consortium was 12.50–13.75 ◦ C (Figure 2), supporting the potential use
of this consortium in phenol degradation in Antarctica during summer periods where the
soil surface temperature may reach up to 19.8 ◦ C [49]. Although not crucial, the presence
of (NH4 )2 SO4 and NaCl, and near-neutral pH had little impact on phenol degradation.
None of the interaction effects between variables were significant. CCD analysis on pure
culture of strain AQ5-06 revealed that the optimum conditions for phenol degradation by
this strain were between 10.0 and 12.5 ◦ C with NaCl concentration range of 0.10–0.13 g/L
and pH 6.5–7.0, with these three, and not (NH4 )2 SO4 concentration, being significant
factors [43]. Optimum conditions for strain AQ5-15 are at a pH range of 7.13–7.50, and
15.0–17.5 ◦ C with interaction between these two factors being significant [44]. In a previous
 the soil surface temperature may reach up to 19.8 °C [49]. Although not crucial, the pres-
ence of (NH4)2SO4 and NaCl, and near-neutral pH had little impact on phenol degradation.
None of the interaction effects between variables were significant. CCD analysis on pure
culture of strain AQ5-06 revealed that the optimum conditions for phenol degradation by
this strain were between 10.0 and 12.5 °C with NaCl concentration range of 0.10–0.13 g/L
Diversity 2021, 13, 643 7 of 19
and pH 6.5–7.0, with these three, and not (NH4)2SO4 concentration, being significant fac-
tors [43]. Optimum conditions for strain AQ5-15 are at a pH range of 7.13–7.50, and 15.0–
17.5 °C with interaction between these two factors being significant [44]. In a previous
study,ANOVA
study, ANOVAofofRSM RSMdatadatafor
forphenol
phenoldegradation
degradationby byaamicrobial
microbialconsortium
consortiumrevealed
revealed
thatpH
that pHandandtemperature
temperaturewere
weresignificant
significantfactors
factorsbut
butthat
thatthe
theinteraction
interactionbetween
betweenthem
them
wasnot
was notsignificant
significant[31].
[31].

Figure2.2.The
Figure Theone-factor
one-factorplot
plotshowing
showingthe
the significant
significant effect
effect of temperature
of temperature on phenol
on phenol degradation
degradation by
by the binary consortium.
the binary consortium.

TheRSM
The RSMmodel
modelwaswasvalidated
validatedusing
usingthethevalues
valuespredicted
predictedfor
foreach
eachparameter.
parameter.This
This
identified no significant difference (t-test, p = 0.0671) between the percentage of phenol
identified no significant difference (t-test, p = 0.0671) between the percentage of phenol
degradation predicted
degradation predicted by
bythe
themodel
model(91.14%)
(91.14%)andandmeasured
measuredexperimentally
experimentallyapproaches
approaches
(93.84%),supporting
(93.84%), supportingthe
thevalidity
validityof
ofthe
themodel.
model.

3.2.
3.2. Effect
EffectofofPhenol
PhenolInitial
InitialConcentration
Concentrationon onConsortium
ConsortiumDegradation
DegradationActivity
Activity
Figure
Figure 3 shows the bacterial growth and degradation of different initial
3 shows the bacterial growth and degradation of different initial concentra-
concentra-
tions
tionsof ofphenol
phenolby bythe
thebinary
binaryconsortium
consortiumunder underthe theoptimized
optimizedconditions
conditionsobtained
obtainedfrom from
CCD.
CCD.The Thestrains,
strains,when
whenin inconsortium,
consortium,had hadthe
theability
abilitytotodegrade
degradephenol
phenolup uptotoananinitial
initial
concentration
concentration of of 1.7
1.7 g/L within168
g/L within 168h.h.When
Whenthe theinitial
initial concentration
concentration was
was increased
increased to
to 1.9
1.9 g/L, the strains started to lose their degradation ability despite growth
g/L, the strains started to lose their degradation ability despite growth being visible. High being visible.
High concentration
concentration of phenol
of phenol hinders
hinders the microbial
the microbial growth growth
and and its metabolic
its metabolic capacity.
capacity. The Thebac-
bactericidal property of phenol allows it to partition into the cellular
tericidal property of phenol allows it to partition into the cellular membrane, thereby membrane, therebydis-
disrupting membrane
rupting membrane function
function andand resulting
resulting in cell
in cell mortality
mortality [50,51].
[50,51]. Strain
Strain AQ5-06AQ5-06in mon-in
monoculture
oculture diddid notnot exhibit
exhibit significant
significant growthatataaphenol
growth phenol concentration
concentration of of 1.25
1.25 g/L
g/L [43],
[43],
whereas
whereas strain AQ5-15 was inhibited at a phenol concentration of 1.7 g/L [44]. Similarto
strain AQ5-15 was inhibited at a phenol concentration of 1.7 g/L [44]. Similar to
these
thesepure
purecultures,
cultures,WenWenetetal.
al.[52]
[52]also
alsoreported
reportedthat strainRhodococcus
thatstrain Rhodococcussp. sp.SKC
SKCshowed
showed
no significant degradation or growth over 15 days at a phenol concentration of 1.5 g/L
due to the inhibitory nature of the target pollutant. The ability of the binary mixture of the
strains studied here to tolerate up to 1.9 g/L initial phenol concentration and to metabolize
phenol at a concentration as high as 1.7 g/L supports the advantage gained from a defined
mixed culture in phenol biodegradation. Similarly, Senthilvelan et al. [53] reported that
a mixed culture of Pseudomonas putida strain Tan-1 and Staphylococcus aureus strain Tan-2
can degrade a high concentration of phenol in a short period of time compared to both
strains individually. It is possible that the metabolic capacity of monocultures may be
limited to certain concentration ranges of substrates (in this case, phenol), but that the use
of consortia broadens their enzymatic capabilities and provides greater capacity to degrade
higher concentrations of phenol [54,55].
 mixed culture in phenol biodegradation. Similarly, Senthilvelan et al. [53] reported that a
mixed culture of Pseudomonas putida strain Tan-1 and Staphylococcus aureus strain Tan-2
can degrade a high concentration of phenol in a short period of time compared to both
strains individually. It is possible that the metabolic capacity of monocultures may be lim-
Diversity 2021, 13, 643 ited to certain concentration ranges of substrates (in this case, phenol), but that the 8use
of 19of
consortia broadens their enzymatic capabilities and provides greater capacity to degrade
higher concentrations of phenol [54,55].

(a) 0.5 g/L 0.7 g/L 0.9 g/L 1.1 g/L 1.3 g/L

1.5 g/L 1.7 g/L 1.9 g/L Control

2.1

Optical density (OD600 nm)

1.8
1.5
1.2
0.9
0.6
0.3
0
0 12 24 36 48 60 72 84 96 108 120 132 144 156 168
Incubation time (hour)

(b) 0.5 g/L 0.7 g/L 0.9 g/L 1.1 g/L 1.3 g/L

1.5 g/L 1.7 g/L 1.9 g/L Control

2.1
Phenol concentration (g/L)

1.8
1.5
1.2
0.9
0.6
0.3
0
0 12 24 36 48 60 72 84 96 108 120 132 144 156 168
Incubation time (hour)

Figure3.3.(a)
Figure (a)Bacterial
Bacterialgrowth
growthand
and(b)
(b)degradation
degradationofofdifferent
differentinitial
initialconcentrations
concentrationsofofphenol
phenolby
by
the binary consortium under optimized conditions (0.40 g/L (NH4)2SO4, 0.13 g/L NaCl, pH 7.25,
the binary consortium under optimized conditions (0.40 g/L (NH4 )2 SO4 , 0.13 g/L NaCl, pH 7.25,
12.50°C). The control (cross mark) shows the initial phenol concentration in the absence of the bac-
12.50◦ C). The control (cross mark) shows the initial phenol concentration in the absence of the
terial strain. Error bars represent the mean ± standard deviation of three replicates.
bacterial strain. Error bars represent the mean ± standard deviation of three replicates.

3.3. Impact of Heavy Metals on Phenol Biodegradation

Metal ions have proven to have significant effects on phenol biodegradation as they
inhibit the activity of catechol dioxygenase which serves as a key enzyme in phenol
metabolism [56,57]. Studies on baseline values for heavy metals in many Antarctic regions
have reported significant levels. A study on King George Island showed the presence of
Hg, Cd, Pb, Zn, Ni, Cu, As, and Cr in benthic samples [58]. Similarly, assessment of soil
from Fildes Peninsula and Ardley Island revealed the presence of significant concentrations
of Pb, Cu, Zn, Hg, and Cd [59]. Lischka et al. [60] reported the presence of Ag, Cd, Hg, Cr,
Pb, Ni, Co, As, Zn, and Cu in different species of Antarctic octopod sampled near Elephant
Island, South Shetland Islands. For the purposes of the current study, Cu, Cr, Zn, Pb, As,
Ni, Al, Co, Ag, Hg, and Cd were chosen as heavy metals of interest. Our previous studies
on Antarctic isolates revealed that these metals negatively affected the biodegradation
processes [61–64].
 tions of Pb, Cu, Zn, Hg, and Cd [59]. Lischka et al. [60] reported the presence of Ag, Cd,
Hg, Cr, Pb, Ni, Co, As, Zn, and Cu in different species of Antarctic octopod sampled near
Elephant Island, South Shetland Islands. For the purposes of the current study, Cu, Cr,
Zn, Pb, As, Ni, Al, Co, Ag, Hg, and Cd were chosen as heavy metals of interest. Our pre-
Diversity 2021, 13, 643 vious studies on Antarctic isolates revealed that these metals negatively affected the
9 ofbio-
19
degradation processes [61–64].

3.3.1. Arthrobacter sp. Strain AQ5-06

3.3.1. Arthrobacter sp. Strain AQ5-06
The heavy metal tolerance of strain AQ5-06 was assessed by evaluating residual phe-
nol The heavy metal
concentration tolerance
in culture of strain
media amendedAQ5-06
withwas assessed
1.0 ppm by evaluating
of various residual
heavy metal ions
phenol concentration in culture media amended with 1.0 ppm of various heavy metal
(Figure 4). ANOVA confirmed that significant differences in both bacterial growth [F11,12
ions (Figure 4). ANOVA confirmed that significant differences in both bacterial growth
= 200.7, p < 0.0001] and phenol degradation [F11,12 = 141158, p < 0.0001] for strain AQ5-06
[F11,12 = 200.7, p < 0.0001] and phenol degradation [F11,12 = 141158, p < 0.0001] for strain
were apparent with exposure to the different metals. Degradation by this strain was not
AQ5-06 were apparent with exposure to the different metals. Degradation by this strain
inhibited by 1.0 ppm of Cu, Zn, Pb, As, Ni, Al, Co, or Cd, with 100% degradation of 0.5
was not inhibited by 1.0 ppm of Cu, Zn, Pb, As, Ni, Al, Co, or Cd, with 100% degrada-
g/L phenol achieved within 120 h. Exposure to Cr led to 83.52% (±0.0042) degradation and
tion of 0.5 g/L phenol achieved within 120 h. Exposure to Cr led to 83.52% (±0.0042)
to Hg led to 11.82% (±0.0021) degradation within 120 h. Exposure to Ag completely inhib-
degradation and to Hg led to 11.82% (±0.0021) degradation within 120 h. Exposure to
ited phenol degradation. Strain AQ5-06 exhibited different growth patterns in response
Ag completely inhibited phenol degradation. Strain AQ5-06 exhibited different growth
to exposure to the different heavy metals, with the highest growth observed in the pres-
patterns in response to exposure to the different heavy metals, with the highest growth
ence of Cu. Tukey’s multiple comparison test confirmed that exposure Cu led to signifi-
observed in the presence of Cu. Tukey’s multiple comparison test confirmed that exposure
cantly greater growth (1.1890 ± 0.0050) than both the control conditions and all other met-
Cu led to significantly greater growth (1.1890 ± 0.0050) than both the control conditions
als tested.
and Growth
all other metalswas inhibited
tested. Growth in was
the presence
inhibitedofinAg
theand was lowest
presence of Aginand
thewas
presence
lowestof
Hg (0.2555 ± 0.0149), and in both cases was significantly lower than in control
in the presence of Hg (0.2555 ± 0.0149), and in both cases was significantly lower than in conditions
and relative
control to all and
conditions other metalstotested.
relative all other metals tested.

Phenol degradation Bacterial growth

100 1.2

Optical density (OD600 nm)

Phenol degradation (%)

80 1.0

0.8
60
0.6
40
0.4
20 0.2

0 0.0

Heavy metals (1.0 ppm)

Figure4.4.Phenol
Figure Phenoldegradation
degradationandand growth
growth of Arthrobacter
of Arthrobacter sp. strain
sp. strain AQ5-06AQ5-06
in theinpresence
the presence of 1.0
of 1.0 ppm
ppm concentrations of different heavy metals. Error bars represent the mean ± standard deviation
concentrations of different heavy metals. Error bars represent the mean ± standard deviation of three
of three replicates.
replicates.

Following the initial screening, phenol degradation by strain AQ5-06 was tested in
the presence of different concentrations of inhibiting metals (Figure 5). For Ag, ANOVA
confirmed that both bacterial growth [F6,7 = 325.80, p < 0.0001] and phenol degradation
[F6,7 = 501.10, p < 0.0001] were significantly influenced by the metal concentration applied.
Inhibition of phenol degradation commenced at an Ag concentration of 0.7 ppm (38.30%
inhibition), whereas at 0.8 ppm inhibition increased to 66.57%. A similar pattern was
apparent in bacterial growth, with strong inhibition becoming apparent at 0.8 ppm. For
Hg, ANOVA again confirmed that both bacterial growth [F5,6 = 767.80, p < 0.0001] and
phenol degradation [F5,6 = 1518, p < 0.0001] were significantly influenced by the metal
concentration applied. Phenol degradation started to show inhibition by Hg at 0.6 ppm
(26.30% inhibition). At higher concentrations, degradation and growth were progressively
reduced, reaching 88.18% inhibition at 1.0 ppm.
 inhibition), whereas at 0.8 ppm inhibition increased to 66.57%. A similar pattern was ap-
parent in bacterial growth, with strong inhibition becoming apparent at 0.8 ppm. For Hg,
ANOVA again confirmed that both bacterial growth [F5,6 = 767.80, p < 0.0001] and phenol
degradation [F5,6 = 1518, p < 0.0001] were significantly influenced by the metal concentra-
Diversity 2021, 13, 643 tion applied. Phenol degradation started to show inhibition by Hg at 0.6 ppm (26.30%10 of 19
inhibition). At higher concentrations, degradation and growth were progressively re-
duced, reaching 88.18% inhibition at 1.0 ppm.

Ag Hg

100 1.0

Optical density (OD600 nm)

Phenol degradation (%)
80 0.8

60 0.6

40 0.4

20 0.2

0 0.0
0.0 0.2 0.4 0.6 0.8 1.0
Heavy metal concentration (ppm)

Figure5.5. Effects
Figure Effects of
ofexposure
exposuretotodifferent concentrations
different concentrationsof Ag andand
of Ag Hg on
Hgphenol degradation
on phenol (solid
degradation
(solid line) and growth (dotted line) of Arthrobacter sp. strain AQ5-06. Error bars represent the±
line) and growth (dotted line) of Arthrobacter sp. strain AQ5-06. Error bars represent the mean
standard
mean deviation
± standard of threeof
deviation replicates.
three replicates.

TheIC
The IC5050 value
value is is the
theconcentration
concentration of ofaasubstance
substancethatthatreduces
reducesan anenzyme’s
enzyme’sactivity
activity
byby50%.
50%.TheTheIC IC5050 of
of these
thesetwo twoheavy
heavymetals
metalswas wasdetermined
determinedusing usingdose-response
dose-responsecurves curves
(Figure6).
(Figure 6). Based
Based on onthethemodelled
modelledcurve,curve, both AgAg
both and HgHg
and exhibited
exhibitedsimilar levels
similar of inhi-
levels of
bition of phenol
inhibition of phenol degradation
degradation with IC50
with ICvalues
50 valuesof 0.75
of and
0.75 and0.74
0.74ppm,
ppm, respectively.
respectively. Cu
Cu is
isknown
knownasasananessential
essentialtrace traceelement,
element,especially
especiallyininaerobic
aerobicorganisms
organismswhere whereititactsactsasasa
acofactor
cofactorfor forenzymes
enzymes that areare
that responsible
responsible for for
diverse redox
diverse reactions
redox [65].[65].
reactions Similarly, other
Similarly,
heavyheavy
other metals are essential
metals for growth
are essential of microorganisms
for growth of microorganisms at trace concentrations
at trace [66]. Con-
concentrations [66].
versely, heavy
Conversely, heavymetalsmetals suchsuch
as Cd, Hg,Hg,
as Cd, andand Pb have no known
Pb have no known biological rolesroles
biological and and
their
bioaccumulation
their bioaccumulation in the in cell
the over timetime
cell over can can
leadlead
to negative impacts
to negative impacts[66,67]. However,
[66,67]. However, de-
despite their
spite their toxicity,Pb
toxicity, PbandandCd Cddiddidnot
notinhibit
inhibitphenol
phenoldegradation
degradationbybythis thisstrain,
strain,similar
similarto
tothethe report
report ofofYooYoo etetal.al. [68].
[68]. TheThe significant
significant inhibitory
inhibitory effects
effects onon phenol
phenol degradation
degradation de-
detected in this study are similar to those reported by Al-Defiery
tected in this study are similar to those reported by Al-Defiery and Reddy [57] who con- and Reddy [57] who
confirmed
firmed that that phenol
phenol degradationby
degradation Rhodococcuspyridinivorans
byRhodococcus pyridinivorans strain GM3 GM3 was wasinhibited
inhibited
byby AgAg and
and Hg Hg at at low concentration.
concentration. The Theinhibitory
inhibitoryeffects
effectsofofAg AgandandHg Hg areare caused
caused by
by their
their ability
ability to to
bind bind to sulfhydryl
to sulfhydryl (-SH)
(-SH) groups
groups of enzymes
of enzymes responsible
responsible for for microbial
microbial me-
metabolism,
tabolism, thereby thereby impeding
impeding enzymatic
enzymatic actions
actions [69].[69].
TheseThese metals
metals causecause disturbance
disturbance in cel-
in cellular functions and trigger oxidative stress in microorganisms
lular functions and trigger oxidative stress in microorganisms [70]. Ag and Hg are gener- [70]. Ag and Hg are
generally recognized as strong inhibitors of phenol degradation
ally recognized as strong inhibitors of phenol degradation with bacteria unable to resist with bacteria unable to
resist their impacts
their impacts [57]. [57].

3.3.2. Arthrobacter sp. Strain AQ5-15

The heavy metal tolerance of strain AQ5-15 was assessed by evaluating residual
phenol concentration in culture media amended with 1.0 ppm of various heavy metal
ions (Figure 7). ANOVA showed that bacterial growth [F11.12 = 727.50, p < 0.0001] was
significantly influenced by exposure to different metals. As phenol degradation was either
0% or 100% ANOVA could not be applied. Tukey’s multiple comparison test showed
that growth under exposure to Cu was greater (1.2785 ± 0.0135) but not significantly
different from the control, and otherwise significantly greater (all p < 0.05) in comparison
with all other heavy metals tested. The growth of this strain was least in the presence of
Ag (0.2675 ± 0.0025) and Hg (0.2065 ± 0.0045) and was moderate in the presence of Cd
(0.503 ± 0.001), although still significantly lower than the control (p < 0.05).
Diversity 2021, 13, 643 11 of 19
Diversity 2021, 13, 643 11 of 19

(a) (b)

Figure 6. Dose-response curves identifying IC50 values for Arthrobacter sp. strain AQ5-06 phenol degradation after 120-h
exposure to different concentrations of (a) Ag and (b) Hg.

3.3.2. Arthrobacter sp. Strain AQ5-15

The heavy metal tolerance of strain AQ5-15 was assessed by evaluating residual phe-
nol concentration in culture media amended with 1.0 ppm of various heavy metal ions
(Figure 7). ANOVA showed that bacterial growth [F11.12 = 727.50, p < 0.0001] was signifi-
cantly influenced by exposure to different metals. As phenol degradation was either 0%
or 100% ANOVA could not be applied. Tukey’s multiple comparison test showed that
growth under exposure to Cu was greater (1.2785 ± 0.0135) but not significantly different
from the control, and otherwise significantly greater (all p < 0.05) in comparison with all
other heavy metals tested. The growth of this strain was least in the presence of Ag (0.2675
Figure
Figure 6.
6. Dose-response
Dose-response curves
curves identifying
± 0.0025) and IC
identifying Hg
IC50 values
values for
50 (0.2065 Arthrobacter
± 0.0045)
for and sp.
Arthrobacter sp. strain
was AQ5-06
moderate
strain inphenol
AQ5-06 degradation
the presence
phenol of Cdafter
degradation 120-h
(0.503
after ± 0.001),
120-h
exposure to different concentrations
although
exposure to different concentrations of (a) Ag
still
of (a) and (b) Hg.
Agsignificantly
and (b) Hg. lower than the control (p < 0.05).

3.3.2. Arthrobacter sp. Strain AQ5-15

Phenol degradation
The heavy metal tolerance Bacterial
of strain AQ5-15 growth by evaluating residual phe-
was assessed
nol concentration
100 in culture media amended with 1.0 ppm of various1.4heavy metal ions

Optical density (OD600 nm)

(Figure 7). ANOVA showed that bacterial growth [F11.12 = 727.50, p < 0.0001] was signifi-
Phenol degradation (%)

1.2
cantly influenced
80 by exposure to different metals. As phenol degradation was either 0%
or 100% ANOVA could not be applied. Tukey’s multiple comparison1.0test showed that
60
growth under exposure to Cu was greater (1.2785 ± 0.0135) but not significantly
0.8 different
from the control, and otherwise significantly greater (all p < 0.05) in comparison with all
0.6
40 metals tested. The growth of this strain was least in the presence
other heavy of Ag (0.2675
0.4
± 0.0025) and Hg (0.2065 ± 0.0045) and was moderate in the presence of Cd (0.503 ± 0.001),
20
although still significantly lower than the control (p < 0.05). 0.2
0 0.0
Phenol degradation Bacterial growth

100 1.4
Optical density (OD600 nm)

Heavy metals (1.0 ppm)

Phenol degradation (%)

1.2
80
1.0
Figure7.7. Phenol
Phenol degradation
degradation and
and growth
growth of of Arthrobacter
Arthrobacter sp. sp.
strainstrain AQ5-15
AQ5-15 in thein the presence of 1.0
Figure 60
ppm concentrations of different heavy metals. Error bars represent the mean 0.8 presence of 1.0 ppm
± standard deviation
concentrations of different heavy metals. Error bars represent the mean ± standard deviation of three
of three
40replicates. 0.6
replicates.
0.4
Different
20
Different concentrations
concentrations ofof inhibiting
inhibiting metals
metals werewere added
added to media
to media withwith 0.5 g/L
0.5 g/L phe-
phenol
0.2
nol to determine the minimum concentration required for inhibition within a 96 h incuba-
to determine the minimum concentration required for inhibition within a 96 h incubation
tion (Figure
(Figure 8). Ag,
08). For For Ag, ANOVA
ANOVA identified
identified a significant
a significant influence
influence onon both
0.0
both bacterialgrowth
bacterial growth
[F5,6 = 1504, p < 0.0001] and phenol degradation [F5,6 = 264.7, p < 0.0001]. Significant inhi-
bition of phenol degradation started to become apparent at Ag concentration of 0.6 ppm
(44.06% inhibition), increasing to 61.20% inhibition at 0.8 ppm. Bacterial growth similarly
Heavy metals (1.0 ppm)
started to be inhibited at 0.6 ppm. For Hg, ANOVA confirmed a significant influence
on both bacterial growth [F5,6 = 3807, p < 0.0001] and phenol degradation [F5,6 = 12230,
p < 0.0001].
Figure Significant
7. Phenol inhibition
degradation of phenol-degradation
and growth became
of Arthrobacter sp. strain apparent
AQ5-15 at Hg concen-
in the presence of 1.0
ppm concentrations of different heavy metals. Error bars represent the mean ±
tration of 0.2 ppm (57.03% inhibition). At a concentration of 0.4 ppm both degradation standard deviation
of three
and replicates.
growth were completely inhibited. For Cd, ANOVA again confirmed a significant
influence on both bacterial growth [F5,6 = 250.8, p < 0.0001] and phenol degradation
[F5,6 Different
= 1959000,concentrations of inhibiting
p < 0.0001]. Phenol metals
degradation were
was added to media
significantly with
inhibited at a0.5
Cdg/L phe-
concen-
nol to determine
tration of 0.2 ppm the(79.88%
minimum concentration
inhibition). required for inhibition
At a concentration within
of 0.4 ppm, a 96 h incuba-
degradation was
tion (Figureinhibited
completely 8). For Ag, ANOVA
although identified
growth a significant
continued, influence
decreasing withon bothCd
higher bacterial growth
concentration.
 0.0001]. Significant inhibition of phenol-degradation became apparent at Hg concentra-
tion of 0.2 ppm (57.03% inhibition). At a concentration of 0.4 ppm both degradation and
growth were completely inhibited. For Cd, ANOVA again confirmed a significant influ-
ence on both bacterial growth [F5,6 = 250.8, p < 0.0001] and phenol degradation [F5,6 =
Diversity 2021, 13, 643 12 of 19
1959000, p < 0.0001]. Phenol degradation was significantly inhibited at a Cd concentration
of 0.2 ppm (79.88% inhibition). At a concentration of 0.4 ppm, degradation was completely
inhibited although growth continued, decreasing with higher Cd concentration. This
shows
This thatthat
shows thisthis
metal only
metal hinders
only thethe
hinders metabolic
metabolicprocess
processand
anddoes
doesnot
not kill
kill the bacteria.
the bacteria.
The growth without the availability of phenol may indicate that this strain utilized
The growth without the availability of phenol may indicate that this strain utilized nutrientsnutri-
ents recycled from dead cells
recycled from dead cells [71]. [71].

Ag Hg Cd

100 1.2

Optical density (OD600 nm)

1.0
Phenol degradation (%)
80
0.8
60
0.6
40
0.4
20 0.2

0 0.0
0.0 0.2 0.4 0.6 0.8 1.0
Heavy metal concentration (ppm)
.
Figure8.8. Effects
Figure Effects of
ofdifferent
differentconcentrations
concentrations of of Ag,
Ag, Hg,
Hg,and
andCd
Cdononphenol
phenoldegradation
degradation(solid
(solid line)
line)
and
andgrowth
growth(dotted line)ofofArthrobacter
(dottedline) Arthrobactersp.
sp.strain
strainAQ5-15.
AQ5-15.Error
Errorbars
barsrepresent
representthe mean±±standard
themean standard
deviationof
deviation ofthree
threereplicates.
replicates.

IC 50 of these
IC50 these heavy
heavy metals
metals was
was determined
determined using using dose-response
dose-response curves
curves (Figure
(Figure 9).
9).
Based
Based on the modelled
modelledcurve,
curve,the
themetal
metal with
with thethe greatest
greatest inhibitory
inhibitory effect
effect on phenol
on phenol deg-
degradation by strain
radation by strain AQ5-15AQ5-15 waswhich
was Cd Cd which
had the had the lowest
lowest IC50ofvalue
IC50 value of 0.06
0.06 ppm, ppm,
followed
followed
by Hg with by Hg
IC50with IC50
of 0.14 of 0.14
ppm andppm and IC
Ag with Ag50with IC50
of 0.70 ppm.of 0.70 ppm. Reduction
Reduction in phenolindegrada-
phenol
degradation
tion rate with rate with increasing
increasing metal concentration
metal concentration is likelyis to
likely to be
be due todue
toxictoeffects
toxic effects on
on meta-
metabolic pathways, although not necessarily to population decline [72],
bolic pathways, although not necessarily to population decline [72], as illustrated by the as illustrated by
the continued growth of strain AQ5-15 at increasing Cd concentration.
continued growth of strain AQ5-15 at increasing Cd concentration. Similarly, Ahmad et Similarly, Ahmad
et
al.al.
[40][40] reported
reported that
that Arthrobacter
Arthrobacter bambusae
bambusae strain
strain AQ5-003
AQ5-003 exhibited
exhibited intolerance
intolerance to
to 0.6
0.6
ppm ppm of Ag
of Ag with
with <50%<50% phenol
phenol degradation
degradation andand to atoCda concentration
Cd concentration of ppm
of 0.2 0.2 ppm
withwith
20%
20% phenol degradation. These heavy metals have cytotoxic effects
phenol degradation. These heavy metals have cytotoxic effects on both structural and per-on both structural
and permeability
meability properties
properties of cell membranes
of cell membranes and organelles,
and organelles, thereby
thereby inhibiting
inhibiting cellular
cellular enzy-
enzymatic activities, in this case the enzyme system responsible for
matic activities, in this case the enzyme system responsible for phenol degradation. phenol degradation.

3.3.3. Binary Consortium

The tolerance of heavy metals by this binary consortium comprising strains AQ5-06
and AQ5-15 was identified by evaluating residual phenol concentration in culture me-
dia with 1.0 ppm of heavy metal (Figure 10). ANOVA confirmed that bacterial growth
[F11,12 = 756.40, p < 0.0001] was significantly influenced by exposure to different metals.
ANOVA could not be applied for phenol degradation because degradation by this consor-
tium was not inhibited by 1.0 ppm Cu, Cr, Zn, Pb, As, Ni, Al, or Co, with 100% degradation
of 0.5 g/L phenol within 48 h, whereas Ag, Hg and Cd completely inhibited degradation.
The growth patterns differed with exposure to the different heavy metals, with the high-
est growth observed in the absence of heavy metals. Tukey’s multiple comparison tests
confirmed that growth under control conditions (1.350 ± 0.0090) was significantly greater
than with any of the metals tested. Growth was completely inhibited by Hg and was
significantly lower than either control or other metals in the presence of Ag (0.240 ± 0.0200)
and Cd (0.4140 ± 0.0055). This consortium’s inhibition pattern was similar to that described
for strain AQ5-15, with Ag, Hg, and Cd being strong inhibitors of phenol degradation.
Cd did not show an inhibitory effect on pure culture of strain AQ5-06; however, when
Diversity 2021, 13, 643 13 of 19

combined with strain AQ5-15, Cd completely inhibited phenol degradation but not growth.
Lu et al. [73] reported similar results, where the effect of metal ions on polychlorinated
biphenyl (PCB) dechlorination by a binary mixture of Dehalococcoides mccartyi strain CG1
and Geobacter lovleyi strain LYY exhibited similar trends with respect to a pure culture of
Diversity 2021, 13, 643 13 of 19
D. mccartyi strain CG1. These data suggest use of a mixed culture does not necessarily have
a positive influence on tolerance of exposure to toxic heavy metals at high concentration.

(a) (b)

(c)

Figure 9.
Figure 9. Dose-response
Dose-response curves
curves indicating
indicating IC
IC50
50 values
values for Arthrobacter sp.
for Arthrobacter sp. strain AQ5-15 phenol
strain AQ5-15 phenol degradation
degradation after
after 96-h
96-h
exposure to different concentrations of (a) Ag, (b) Hg, and (c)
exposure to different concentrations of (a) Ag, (b) Hg, and (c) Cd.Cd.

3.3.3. Different
Binary Consortium
concentrations of inhibiting heavy metals were added to media with 0.5 g/L
phenol to determine
The tolerance of the starting
heavy concentration
metals of inhibition
by this binary consortiumover acomprising
48 h incubation (Figure
strains 11).
AQ5-06
For Ag,
and ANOVA
AQ5-15 was confirmed
identified bythatevaluating
both bacterial growth
residual phenol = 2296, p < 0.0001]
[F5,6concentration and phenol
in culture media
degradation
with 1.0 ppm[Fof = 14477,
5,6heavy p < (Figure
metal 0.0001] were significantly
10). ANOVA influenced
confirmed thatby metal concentration.
bacterial growth [F11,12
=Inhibition
756.40, p <of0.0001]
phenolwas degradation by influenced
significantly the mixed culture
by exposurestarted
to at an Ag concentration
different metals. ANOVA of
0.2 ppm (48.64% inhibition) and further increase in concentration
could not be applied for phenol degradation because degradation by this consortium was resulted in complete
inhibition.
not inhibitedBacterial
by 1.0 growth
ppm Cu, showed
Cr, Zn,a similar
Pb, As, response
Ni, Al, ortoCo, increasing
with 100% concentration.
degradation For
ofHg,
0.5
ANOVA again confirmed that both bacterial
g/L phenol within 48 h, whereas Ag, Hg and Cd completely growth [F 6,7 = 17518, p < 0.0001] and phenol
inhibited degradation. The
degradation
growth [F6,7 differed
patterns p < 0.0001]
= 45824, with weretosignificantly
exposure the different influenced by metal
heavy metals, concentration.
with the highest
growth observed in the absence of heavy metals. Tukey’s multiple comparison tests At
Inhibition of degradation started at a concentration of 0.5 ppm (58.25% inhibition). a
con-
concentration of 0.6 ppm, inhibition reached 89.76% and further increase
firmed that growth under control conditions (1.350 ± 0.0090) was significantly greater than in concentration
resulted
with anyinofcomplete
the metals inhibition. For Cd, was
tested. Growth ANOVA also confirmed
completely inhibitedthat
byboth bacterial
Hg and was growth
signifi-
[F6,7 = 17086, p < 0.0001] and phenol degradation [F 6,7 = 16423, p < 0.0001]
cantly lower than either control or other metals in the presence of Ag (0.240 ± 0.0200) and were significantly
influenced by metal concentration. Inhibition of degradation started at Cd concentration
Cd (0.4140 ± 0.0055). This consortium’s inhibition pattern was similar to that described for
of 0.8 ppm (48.81% inhibition). At 0.9 ppm inhibition reached 89.76%. In this case, how-
strain AQ5-15, with Ag, Hg, and Cd being strong inhibitors of phenol degradation. Cd
did not show an inhibitory effect on pure culture of strain AQ5-06; however, when com-
bined with strain AQ5-15, Cd completely inhibited phenol degradation but not growth.
Lu et al. [73] reported similar results, where the effect of metal ions on polychlorinated
biphenyl (PCB) dechlorination by a binary mixture of Dehalococcoides mccartyi strain CG1
and Geobacter lovleyi strain LYY exhibited similar trends with respect to a pure culture of
Diversity 2021, 13, 643 14 of 19

Diversity 2021, 13, 643 14 of 19

ever, growth was strongly inhibited, having initially increased slightly when exposed to
concentrations of up 0.4 ppm before decreasing with further increase in concentration.

Phenol Degradation Bacterial Growth

100 1.5

Optical density (OD600 nm)

Phenol degradation (%) 80 1.2

60 0.9

40 0.6

20 0.3

0 0.0

Heavy metals (1.0 ppm)

Figure 10.
Figure 10. Effects
Effectsof of
1.01.0
ppm concentration
ppm of different
concentration heavy metals
of different heavy on phenol
metals ondegradation and growth
phenol degradation
Diversity 2021, 13, 643 on the binary consortium. Error bars represent the mean ± standard deviation of three 15 of 19
replicates.
and growth on the binary consortium. Error bars represent the mean ± standard deviation of
three replicates.
Different concentrations of inhibiting heavy metals were added to media with 0.5 g/L
phenol to determine the starting concentration of inhibition over a 48 h incubation (Figure 11).
For Ag, ANOVA confirmed that Agboth bacterial
Hg Cd [F5,6 = 2296, p < 0.0001] and phenol
growth
degradation
100 [F5,6 = 14477, p < 0.0001] were significantly influenced by metal 1.8 concentration.
Inhibition of phenol degradation by the mixed culture started at an Ag concentration of

Optical density (OD600 nm)

Phenol degradation (%)

0.2 ppm80(48.64% inhibition) and further increase in concentration resulted 1.5 in complete
inhibition. Bacterial growth showed a similar response to increasing concentration. For
Hg, ANOVA60 again confirmed that both bacterial growth [F6,7 = 17518,1.2 p < 0.0001] and phe-
nol degradation [F6,7 = 45824, p < 0.0001] were significantly influenced0.9 by metal concentra-
tion. Inhibition
40 of degradation started at a concentration of 0.5 ppm (58.25% inhibition).
At a concentration of 0.6 ppm, inhibition reached 89.76% and further0.6 increase in concen-
tration resulted
20 in complete inhibition. For Cd, ANOVA also confirmed that both bacterial
0.3
growth [F6,7 = 17086, p < 0.0001] and phenol degradation [F6,7 = 16423, p < 0.0001] were
significantly
0 influenced by metal concentration. Inhibition of degradation 0.0 started at Cd
concentration0.0 of 0.8 ppm
0.2 (48.81%0.4 inhibition).
0.6 At 0.9 ppm
0.8 inhibition
1.0 reached 89.76%. In
this case, however, growth was strongly inhibited, having initially increased slightly
Heavy metal concentration (ppm)
when exposed to concentrations of up 0.4 ppm before decreasing with further increase in
concentration.
Figure
Figure The
11.pure
11. The cultures of
The influence
influence of strains
ofexposure
exposureAQ5-15 andconcentrations
totodifferent
different AQ5-06 couldoftolerate
concentrations Ag,Ag,
of HgHgAg concentrations
andand
Cd Cd
on phenol up
deg-
on phenol
radation
to 0.4 and(solid
0.6 line)
ppm, and the growth
respectively, (dotted
whereas line)
theof the
binarybinary consortium.
mixture
degradation (solid line) and the growth (dotted line) of the binary consortium. could only tolerate up to 0.2
ppm, at which concentration phenol degradation was already reduced to half of the con-
trol The
level.pure
Thiscultures of strainswith
result contrasts AQ5-15 and AQ5-06
a number could
of studies tolerategreater
reporting Ag concentrations
resistance of
(a) up to 0.4 and 0.6 ppm, (b) whereas the binary mixture could only tolerate up to
respectively,
mixed microbial cultures compared with pure cultures towards toxic heavy metals [74,75].
0.2 ppm, atthis
However, which concentration
mixture phenol degradation
showed increased tolerance to was higheralready reduced to
concentrations of half of the
Hg and Cd
control level. This result contrasts with a number of studies reporting
than did pure culture of strain AQ5-15, possibly suggesting that strain AQ5-06 may have greater resistance of
mixed microbial cultures compared with pure cultures towards toxic heavy
a positive synergistic impact on its co-culture’s tolerance. IC50 of these heavy metals was metals [74,75].
However,
determined thisfrom
mixture showed increased
dose-response tolerance
curves (Figure 12).toBased
higheron concentrations
the modelledofcurve, Hg andAg
Cd than did pure culture of strain AQ5-15, possibly suggesting that
showed the strongest inhibition of phenol degradation with the lowest IC50 value of 0.15 strain AQ5-06 may
have
ppm,a followed
positive synergistic
by Hg withimpact
an IC50onofits co-culture’s
0.48 ppm and Cd tolerance.
with anICIC 5050of
ofthese heavy metals
0.80 ppm.
was determined from dose-response curves (Figure 12). Based on the modelled curve,
Ag showed the strongest inhibition of phenol degradation with the lowest IC50 value of
0.15 ppm, followed by Hg with an IC50 of 0.48 ppm and Cd with an IC50 of 0.80 ppm.
 0 0.0
0.0 0.2 0.4 0.6 0.8 1.0
Heavy metal concentration (ppm)

Diversity 2021, 13, 643 15 of 19

Figure 11. The influence of exposure to different concentrations of Ag, Hg and Cd on phenol deg-
radation (solid line) and the growth (dotted line) of the binary consortium.

(a) (b)

(c)

Figure 12. Dose-response curves indicating IC50 50 values

values for
for the
the binary
binary consortium
consortium phenol
phenol degradation
degradation after 48-h exposure
to different concentrations of (a) Ag, (b) Hg, and (c) Cd.
to different concentrations of (a) Ag, (b) Hg, and (c) Cd.

4.
4. Conclusions
Conclusions
This
This study utilized
utilized the
the advantage
advantageof
ofstatistical
statisticalexperimental
experimentaldesign
design
toto optimize
optimize phe-
phenol
nol biodegradation by a binary consortium of native Antarctic bacteria. The
biodegradation by a binary consortium of native Antarctic bacteria. The outcome sheds outcome
light on the potential application of RSM in the modeling and optimization of environmen-
tal factors in order to improve phenol degradation. Under optimized conditions, the mixed
culture had better tolerance to a high concentration of phenol compared to pure culture.
In terms of heavy metal tolerance, the binary consortium did not present any advantage
over pure culture at high concentrations. Hence, further studies are needed to ensure the
effectiveness of this binary consortium to bioremediate phenol pollution in the presence
of different co-contaminants. The data from this study provides a solid foundation that
will support the development of practical field bioremediation strategies, particularly in
wastewater treatment systems in the Antarctic environment.

Author Contributions: Conceptualization, S.A.A., P.C., A.Z. and N.A.S.; methodology, A.Z., S.A.A.,
K.S. and K.A.K.; software, K.S. and K.A.K.; validation, K.S., A.Z., S.A.A., P.C., N.A.S. and K.A.K.;
formal analysis, K.S., A.Z., S.A.A. and K.A.K.; investigation, K.S.; resources, S.A.A. and C.G.-F.;
original draft preparation, K.S.; Writing—review and editing S.A.A., P.C., N.A.S., K.A.K., T.A.T.-M.,
C.G.-F. and A.Z.; supervision, S.A.A., P.C., N.A.S., K.A.K. and A.Z.; project administration, S.A.A.,
P.C., A.Z. and C.G.-F.; funding acquisition, A.Z., S.A.A. and C.G.-F. All authors have read and agreed
to the published version of the manuscript.
Funding: This project was financially supported by Shibaura Institute of Technology, Japan under
Research Exchange Program Academic Year 2019 attached to K. Subramaniam, Matching Grant
Diversity 2021, 13, 643 16 of 19

PUTRA (UPM-YPASM 9300430), PUTRA-IPS (9631800), PUTRA-Berimpak (9660000) disbursed by

Universiti Putra Malaysia (UPM) and YPASM Berth Support by Sultan Mizan Antarctic Research
Foundation (YPASM) fund under the research grant attached to S.A. Ahmad. P. Convey is supported
by NERC core funding to the BAS ‘Biodiversity, Ecosystems and Adaptation’ Team. C. Gomez-
Fuentes is supported by Centro de Investigacion y Monitoreo Ambiental Antàrctico (CIMAA) Project.
K. Subramaniam is funded by a Graduate Research Fellowship (GRF) from Universiti Putra Malaysia.
Institutional Review Board Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors would like to thank Shibaura Institute of Technology, Universiti
Putra Malaysia, Centro de Investigacion y Monitoreo Ambiental Antàrctico (CIMAA), Sultan Mizan
Antarctic Research Foundation (YPASM) and National Antarctic Research Centre (NARC).
Conflicts of Interest: The authors declare no conflict of interest.

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